Exhale and Decell: Environmentally Sustainable Sterilization and Decellularization of Cartilage Grafts via Supercritical Carbon Dioxide

Abstract

PURPOSE: Current decellularization techniques have important limitations, including obligatory use of harsh ionized substances or detergents that deplete bioactive cytokines and compromise the physical integrity of the resulting extracellular matrix (ECM) scaffold. Furthermore, the detergents used are increasingly recognized as harmful to the environment and are poised to be restricted from commercial use. Supercritical carbon dioxide (scCO2) is an appealing alternative, applying the principles behind supercritical fluid extraction and sterilization to the removal of immunogenic cellular components. We endeavor to evaluate the efficacy of scCO2 in both sterilization and decellularization of ovine costal cartilage xenograft and human costal cartilage allograft to develop a highly efficacious, environmentally sustainable, and biocompatible alternative to currently available decellularization strategies. METHODS AND MATERIALS: Xenograft preparation: Racks of lamb were purchased from a local butcher. Floating ribs were minced into ∼8 mm3 cubes or zested into flakes (1 mm × 1 mm). Allograft harvest: Human costal cartilage was obtained from patients undergoing microsurgical breast reconstruction necessitating partial rib resection and processed via mincing or zested as above. All samples were subjected to: 1) sterilization; 2) sterilization and decellularization; or 3) sterilization, decellularization, and a pretreatment wash. Sterilization: Samples were placed in a NovaSterilis ™ Nova2200 unit and subject to standard NovaSterilis ™ sterilization parameters. At least 3 samples with biologic indicators were placed within the packaging and processed with each run. Decellularization: Samples were subject to the standard parameters of the Nova2200 system as previously described by NovaSterilis ™ with an ethanol to scCO2 volume ratio of 1:3.3. Wash: Samples were serially soaked in saline and agitated with exposure to 16 mL 3% H2O2 and a 30-minute scCO2 run at 35° C and 1,436 psi. Histology: After treatment, H&E, DAPI, and safranin-O staining were performed. DNA Quantification: DNA content was quantified in unprocessed and decellularized graft samples using the DNeasy Blood & Tissue kit (Qiagen Inc.). RESULTS: Sterilization conditions were sufficient for a 6 log10 sterilization of B. atrophaeus. H&E staining revealed preservation of tissue architecture after both sterilization and decellularization in both allograft and xenograft cartilage. DAPI staining demonstrated depletion of nuclei in the decellularized zested samples, but persistence of visible nuclei in the minced samples. Safranin-O staining revealed immunogenic GAG depletion after decellularization. DNA content in minced and zested samples was 192.2 ng DNA/mg tissue and 321.6 ng DNA/mg tissue, respectively, while DNA content in sterilized and decellularized minced and zested samples were 24.8 ng DNA/mg tissue and 11.6 ng DNA/mg tissue, respectively (industry standard requirement for decellularization <50 ng DNA/mg tissue); after sterilization, decellularization, and the chemical wash, minced and zested samples contained 17.6 ng DNA/mg tissue and 4 ng DNA/mg tissue respectively. CONCLUSION: These preliminary data suggest that scCO2 sufficiently sterilizes and decellularizes cartilage derived from both human and animal sources, thereby supporting scCO2 as an efficacious, commercially appealing, and ecologically responsible alternative to current decellularization strategies. Biomechanical testing on treated samples, as well as the evaluation of scCO2 as a decellularization agent for human nasal septal cartilage are ongoing.